Angewandte
Communications
Chemie
bioreduction and the cascade halted at the ketone intermedi-
ate (entry 4). Regarding the temperature, an increase to 458C
did not impact the outcome of the process (entry 5). More-
over, in an effort to drive the bioreduction of the ketone to
completion, the amount of NADPH was increased to 10 mm
(entry 6). However, the overall conversion was again anch-
ored at the previous 85%. Accordingly, we ran identical
reaction in the absence of NADPH and, surprisingly, the
process worked just as well as in presence of the cofactor
(entry 7).[15] A plausible explanation would be that the
enzyme powder may contain cofactor from the manufacturing
process, according to literature from the supplier of
KREDs.[22,23] On the basis of these findings, we hypothesized
that the reason for not reaching quantitative conversion in the
concurrent approach lies in the stability of the biocatalyst.
Hence, the KRED would be rapidly deactivated in the
reaction medium, thus leading to a fixed conversion of 85%.
In the sequential approach, on the contrary, accumulation of
ketone prior to addition of biocatalyst would enable complete
reduction before enzyme inactivation. To demonstrate these
assumptions, we studied the kinetics of the reactions in more
detail. First, the allylic isomerization of 4a into 4b was
assayed under the reaction conditions of the concurrent
process, namely 5 mol% catalyst loading at 308C (entry 2).
Thus, these milder reaction conditions demanded a longer
reaction time (4 h) for complete conversion, despite using
5 mol% of 2 (see Figure S1 in the Supporting Information).
Likewise, the bioreduction of 4b, catalyzed by KRED-P1-
A04 (Table 1, entry 1), was monitored periodically and 4b
was almost completely reduced in just 30 minutes (90%),
then taking more 20 hours for consumption of the substrate
(see Figure S2). Hence, the reaction slowed down drastically
after 30 minutes, perhaps because of the lack of robustness of
the biocatalyst. Alternatively, this enzymatic reaction was also
performed but the KRED in the buffer was incubated for
3 hours prior to the addition of 4b. Now, the bioreduction
reached 85% conversion very quickly, although it did not
evolve further. Indeed, this outcome was identical to that
achieved in the concurrent process (Table 3, entry 2), in which
4b is gradually formed by the action of 2 while the enzyme
decomposed. Finally, the distribution of products of the
concurrent process (conversion of 4a into 4c) was checked
over time (entry 2), thus showing that the overall trans-
formation occurs mainly in the first 4 hours, a period during
which the enzyme remains highly active (Figure 1). Collec-
tively, these facts indicate that the faster the isomerization
takes place, the higher the overall yield of the cascade.
Bearing this information in mind, the concurrent approach
was extended to the allylic alcohols 6a and 9a with the same
catalytic system (2 and KRED-P1-A04, without cofactor).
Pleasantly, the outcome agreed with our expectations, and
furnished enantiopure 6c and 9c in 94 and 91% conversion
(free of unsaturated alcohol), respectively (entries 8 and 9).
On the contrary, the slower isomerization of 5a consequently
brought a moderate overall conversion of 5c (entry 10).
In summary, we have developed a chemoenzymatic one-
pot process in aqueous medium, in both sequential and
concurrent fashion, and it provides enantiomerically pure
alcohols from racemic allylic alcohols by combining
Figure 1. Product distribution as a function of time for the conversion
of 4a into 4c concurrently catalyzed by 2 and KRED-P1-A04 (see
Table 3, entry 2 for reaction conditions).
a ruthenium(IV)-catalyzed isomerization with a bioreduction
promoted by KREDs. The sequential process was highly
efficient and operationally simple, the only adjustment before
KRED and cofactor addition being a slight decrease on the
temperature. Regarding the concurrent process, both the
metal catalyst and biocatalyst were able to coexist and work
simultaneously from the beginning, thus furnishing the final
products with yields close to 85%. Clearly, this first example
of a genuine concurrent metal-catalyzed and biocatalyzed
reaction in water is an important contribution to the field of
cascade processes, and opens up a new frontier for its practical
application, as it does not require the isolation or compart-
mentalization of the catalysts.
Acknowledgments
We are indebted to the MINECO of Spain (RYC-2011-08451
and CTQ2013-40591-P), the Gobierno del Principado de
Asturias (Project GRUPIN14-006), and the COST Action
SIPs-CM1302 for financial support. J.G.-A. thanks the
MINECO and the European Social Fund for a “Ramꢁn
y Cajal” contract. E.L. acknowledges funding from the
European Unionꢂs Horizon 2020 MSCA ITN-EID program
(grant agreement No 634200). N.R.-L. acknowledges
MINECO for funding under Torres-Quevedo program
(PTQ-12-05 407).
Keywords: alcohols · chirality · enzyme catalysis ·
isomerization · transition metals
[1] P. T. Anastas, J. C. Warner, Green Chemistry Theory and
Practice, Oxford University Press, Oxford, 1998.
[2] a) U. M. Lindstrçm, Organic Reactions in Water: Principles,
Strategies and Applications, Blackwell Publishing, Oxford, 2007;
4
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!